1. Field of the Invention
The present invention relates to the field of molecular biology. The invention discloses compositions comprising the nucleotide sequence of Mycoplasma genitalium, fragments thereof, and its use in medical diagnostics, therapies and pharmaceutical development.
2. Related Background Art
Mycoplasmas are the smallest free-living bacterial organisms known (Colman, S. D. et al., Mol. Microbiol. 4:683-687 (1990)). Mycoplasmas are thought to have evolved from higher gram-positive bacteria through the loss of genetic material (Bailey, C. C. et al., J. Bacteriol. 176:5814-5819 (1994)). Mycoplasma genitalium (M. genitalium)is widely considered to be the smallest self-replicating biological system, as the molecular size of its genome has been shown to be only 570-600 kp (Pyle, L. E. et al., Nucleic Acids Res. 16(13):6015-6025 (1988); Peterson, S. N. et al., J. Bacteriol. 175:7918-7930 (1993)). All mycoplasmas lack a cell wall and have small genomes and a characteristically low G+C content (Razin, S., Microbiol. Rev. 49(4):419-455 (1985); Peterson, S. N. et al., J. Bacteriol. 175:7918-7930 (1993)). Some mycoplasmas, including M genitalium, have a specialized codon usage, whereby UGA encodes tryptophan rather than serving as a stop codon (Inamine, J. M. et al., J. Bacteriol. 172:504-506 (1990); Tanaka, J. G. et al., Nucleic Acids Res. 19:6787-6792 (1991); Yamao, F. A. et al., Proc. Natl. Acad. Sci. USA 82:2306-2309 (1985)).
Mycoplasmas are widely known to be significant pathogens of humans, animals, and plants (Bailey, C. C. et al., J. Bacteriol. 176:5814-5819 (1994)). The metabolic systems of mycoplasmas indicate that they are generally biosynthetically deficient, and thus depend on the microenvironment of the host by characteristically adhering to host cells in order to obtain essential precursor molecules, i.e., amino acids, fatty acids and sterols etc. (Baseman, J. B., 1987. Micoplasma Membranes, Vol. 20. The Plenum Press, New York, N.Y.).
In particular, M. genitalium, a newly discovered species, is a pathogenic etiological agent first isolated in 1980 from the urethras of human males infected with non-gonococcal urethritis (Tully, J. G. et al., Lancet 1:1288-1291 (1981); Tully, J. G., et al., Int. J. Syst. Bacteriol. 33:387-396 (1983)). M. genitalium has also been identified in specimens of pneumonia patients as a co-isolate of Micoplasma pneumoniae (Baseman, J. B. et al., J. Clin. Microbiol. 26:2266-2269 (1988)). M. genitalium opportunistic infection has often been observed in individuals infected with human immunodeficiency virus type 1 (HIV-1) (Lo, S.-C. et al., Amer. J. Trop. Med. Hyg. 41:601-616 (1989); Lo, S.-C. et al., Amer. J. Trop. Med. Hyg. 41:601-616 (1989); Sasaki, Y. et al., AIDS Res. Hum. Retrov. 9(8):775-780 (1993)). Mycoplasmas can also induce various cytokines, including tumor necrosis factor, which may enhance HIV replication (Chowdhury, I. H. et al., Biochem. Biophys. Res. Commun. 170:1365-1370 (1990)).
A high amino acid homology exists between the attachment protein of M. genitalium and the aligned proteins of several human Class II major histocompatibility complex proteins (HLA), suggesting that M. genitalium infection may play an important role in triggering autoimmune mechanisms, thereby aggravating the immunodeficiency characteristics of acquired immune deficiency syndrome (AIDS) (Montagnier, L. et al., C.R. Acad. Sci. Paris 311(3):425-430 (1990); Root-Bernstein, R. S. et al., Res. Immunol. 142:519-523 (1991); Bisset, L. R. Autoimmunity 14:167-168 (1992)). A diagnostic immunoassay for detecting M. genitalium infection using monoclonal antibodies specific for some M. genitalium antigens has been developed. Baseman, J. B. et al., U.S. Pat. No. 5,158,870.
Due to its diminutive genomic size, M. genitalium provides a useful model for determining the minimum number of genes and protein products necessary for a host-independent existence. M. genitalium expresses a characteristically low number of base-pairs and low G+C content, which along with its UGA tryptophan codon, has hampered sequencing efforts by conventional techniques (Razin, A., Microbiol. Rev. 49(4):419-455 (1985); Colman, S. D. et al., Gene 87:91-96 (1990); Dybvig, K. 1992. Gene Transfer In: Maniloff, J. (ed.) Mycoplasmas: Molecular Biology and Pathogenesis., Am. Soc. Microbiol. Washington, D.C., pp.355-362)). M. genitalium possesses a single circular chromosome (Colman, S. D. et al., Gene 87:91-96 (1990); Peterson, S. N. et al., J. Bacteriol. 175:7918-7930 (1993)). The characterization of the genome of M. genitalium has also been hampered by the lack of auxotrophic mutants and by the lack of a system for genetic exchange, precluding reverse genetic approaches. Thus, the sequencing of the M. genitalium genome would enhance the understanding of how M. genitalium causes or promotes various invasive or immunodeficiency diseases and to how best to medically combat mycoplasma infection.
Prior attempts at characterizing the structure and gene arrangement of the chromosomes of mycoplasmas using pulsed-field gel electrophoretic methods (Pyle, L. E. et al., Nucleic Acids Res. 16(13):6015-6025 (1988); Neimark, H. C. et al., Nucleic Acids Res. 18(18):5443-5448 (1990)), indicated that mycoplasmas have genomes ranging widely in size. Southern blot hybridization of digested DNAs of M. genitalium compared to the well-known human pathogen, M. pneumoniae, indicated overall low homology values of approximately 6-8% (Yogev, D. et al., Int. J. Syst. Bacteriol. 36(3):426-430 (1986)). However, high homologies have been reported between the adhesin genes of M. genitalium and M pneumoniae (Dallo, S. F. et al., Microbial Path. 6:69-73 (1989)). Initial studies at characterizing the genome of M. genitalium by comparison to the well-known M. pneumoniae species, indicated that both species have three (3) rRNA genes clustered together in a chromosomal segment of about 5 kb and form a single operon organized in classical procaryotic fashion, but differences exist between their respective restriction sites (Yogev, D. et al., Int. J. Bacteriol. 36(3):426-430 (1986)).
Restriction enzyme mapping of M. genitalium indicates that the genome is approximately 600 kb. Several genes have also been mapped, including the single ribosomal operon, and the gene encoding the MgPa cytadhesion protein (Su, C. J. et al., J. Bacteriol. 172:4705-4707 (1990); Colman, S. D. et al., Mol. Microbiol. 4(4):683-687 (1990)). The entire restriction map of the genome of M. genitalium has also been cloned in an ordered library of 20 overlapping cosmids and one xcex clone (Lucier, T. S. et al., Gene 150:27-34 (1994)).
An initial study using random sequencing techniques to characterize the M. genitalium genome resulted in forty-four (44) random clones being partially sequenced; several long open reading frames were also found (Peterson, S. N. et al., Nucleic Acids Rev. 19:6027-6031 (1991)). Subsequent work using random sequencing of 508 random nonidentical clones has allowed sequence information to be compiled for approximately seventeen percent (17%) (100,993 nucleotides) of the M. genitalium genome (Peterson, S. N. et al., J. Bacteriol. 175:7918-7930 (1993)). Sequence information indicates that the diminutive genome of M. genitalium contains numerous genes involved in various metabolic processes. The genome is estimated to encode approximately 390 proteins, indicating that M. genitalium makes very efficient use of its limited amount of DNA (Peterson, S. N. et al., J. Bacteriol. 175:7918-7930 (1993)).
Several studies have been undertaken to sequence and characterize individual genes identified in M. genitalium. In particular, the medically important aspects of M. genitalium have helped to direct interest to those genes which determine the degree of infectivity and the virulence characteristics of the organism. The nucleotide sequence and deduced amino acid sequence for the MgPa adhesin gene, ie., the gene encoding the surface cytadhesion protein of M. genitalium, indicates that the complete gene contains 4,335 nucleotides coding for a protein of 159,668 Da. (Dallo, S. F. et al., Infect. Immun. 57(4):1059-1065 (1989)). Furthermore, subsequent nucleotide sequencing of the M. genitalium MgPa adhesin gene revealed the specific codon order for this important gene (Inamine, J. M. et al., Gene 82:259-267 (1989)). The MgPa adhesin gene also has been shown to express restriction fragment length polymorphism (Dallo, S. F. et al., Microbial Path. 10:475-480 (1991)). Nucleotide homology to the well-known highly conserved procaryotic origin-of-replication gene (gyrA) was noted for M. genitalium (Bailey, C. C. et al., J. Bacteriol. 176:5814-5819 (1994)). The highly conserved procaryotic elongation factor, Tu, encoded by the tuf gene, has been noted and sequenced for M. genitalium, and was found to contain an open reading frame encoding a protein of approximately 393 amino acids (Loechel, S. et al., Nucleic Acids Res. 17(23):10127 (1989)). The tuf gene of M. genitalium has also been determined to use a signal other than a Shine-Delgamo (ribosomal binding site) sequence preceding the initiation codon (Loechel, S. et al., Nucleic Acids Res. 19:6905-6911 (1991)).
The present invention is based on the sequencing of the Mycoplasma genitalium genome. The primary nucleotide sequence which was generated is provided in SEQ ID NO:1.
The present invention provides the generated nucleotide sequence of the Mycoplasma genitalium genome, or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan. In one embodiment, present invention is provided as a contiguous string of primary sequence information corresponding to the nucleotide sequence depicted in SEQ ID NO:1.
The present invention further provides nucleotide sequences which are at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1.
The nucleotide sequence of SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence which is at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1 may be provided in a variety of mediums to facilitate its use. In one application of this embodiment, the sequences of the present invention are recorded on computer readable media. Such media includes, but is not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
The present invention further provides systems, particularly computer-based systems which contain the sequence information herein described stored in a data storage means. Such systems are designed to identify commercially important fragments of the Mycoplasma genitalium genome.
Another embodiment of the present invention is directed to isolated fragments of the Mycoplasma genitalium genome. The fragments of the Mycoplasma genitalium genome of the present invention include, but are not limited to, fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of Mycoplasma genitalium in a sample, hereinafter, diagnostic fragments (DFs).
Each of the ORF fragments of the Mycoplasma genitalium genome disclosed in Tables 1(a), 1(c) and 2, and the EMF found 5xe2x80x2 to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers for the presence of a specific microbe in a sample, for the production of commercially important pharmaceutical agents, and to selectively control gene expression.
The present invention further includes recombinant constructs comprising one or more fragments of the Mycoplasma genitalium genome of the present invention. The recombinant constructs of the present invention comprise vectors, such as a plasmid or viral vector, into which a fragment of the Mycoplasma genitalium has been inserted.
The present invention further provides host cells containing any one of the isolated fragments of the Mycoplasma genitalium genome of the present invention. The host cells can be a higher eukaryotic host such as a mammalian cell, a lower eukaryotic cell such as a yeast cell, or can be a procaryotic cell such as a bacterial cell.
The present invention is further directed to isolated proteins encoded by the ORFs of the present invention. A variety of methodologies known in the art can be utilized to obtain any one of the proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. In an alternative method, the protein is purified from bacterial cells which naturally produce the protein. Lastly, the proteins of the present invention can alternatively be purified from cells which have been altered to express the desired protein.
The invention further provides methods of obtaining homologs of the fragments of the Mycoplasma genitalium genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention. Specifically, by using the nucleotide and amino acid sequences disclosed herein as a probe or as primers, and techniques such as PCR cloning and colony/plaque hybridization, one skilled in the art can obtain homologs.
The invention further provides antibodies which selectively bind one of the proteins of the present invention. Such antibodies include both monoclonal and polyclonal antibodies.
The invention further provides hybridomas which produce the above-described antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
The present invention further provides methods of identifying test samples derived from cells which express one of the ORF of the present invention, or homolog thereof. Such methods comprise incubating a test sample with one or more of the antibodies of the present invention, or one or more of the DFs of the present invention, under conditions which allow a skilled artisan to determine if the sample contains the ORF or product produced therefrom.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the above-described assays.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the antibodies, or one of the DFs of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of bound antibodies or hybridized DFs.
Using the isolated proteins of the present invention, the present invention further provides methods of obtaining and identifying agents capable of binding to a protein encoded by one of the ORFs of the present invention. Specifically, such agents include antibodies (described above), peptides, carbohydrates, pharmaceutical agents and the like. Such methods comprise the steps of:
(a) contacting an agent with an isolated protein encoded by one of the ORFs of the present invention; and
(b) determining whether the agent binds to said protein.
The complete genomic sequence of M. genitalium will be of great value to all laboratories working with this organism and for a variety of commercial purposes. Many fragments of the Mycoplasma genitalium genome will be immediately identified by similarity searches against GenBank or protein databases and will be of immediate value to Mycoplasma researchers and for immediate commercial value for the production of proteins or to control gene expression. A specific example concerns PHA synthase. It has been reported that polyhydroxybutyrate is present in the membranes of M. genitalium and that the amount correlates with the level of competence for transformation. The PHA synthase that synthesizes this polymer has been identified and sequenced in a number of bacteria, none of which are evolutionarily close to M. genitalium. This gene has yet to be isolated from M. genitalium by use of hybridization probes or PCR techniques. However, the genomic sequence of the present invention allows the identification of the gene by utilizing search means described below.
Developing the methodology and technology for elucidating the entire genomic sequence of bacterial and other small genomes has and will greatly enhance the ability to analyze and understand chromosomal organization. In particular, sequenced genomes will provide the models for developing tools for the analysis of chromosome structure and function, including the ability to identify genes within large segments of genomic DNA, the structure, position, and spacing of regulatory elements, the identification of genes with potential industrial applications, and the ability to do comparative genomic and molecular phylogeny.